PROJECTSUMMARY Brain cells exhibit profound molecular and cellular changes during aging. Epigenomic marks such as DNA methylationareassociated withage in multiplehuman tissues, suggestingan alteration of transcriptional regulationduringaging. However, age-associated epigenomic signatures have not beendetermined with cell- type specificity in the brain. Single-cell epigenomic strategies, such as single-cell DNA methylation and open chromatin profiling assays, are powerful strategies for de novo identification of cell-type specific epigenome landscapesinheterogeneoustissues.Atthesametime,thesestrategiesuniquelyallowtheidentificationofcell- typespecificregulatoryelementsthatcontrolgeneexpressionpatternsincomplextissues.Theproposedproject will complement and build upon current NIH-supported BRAIN Initiative efforts to produce an epigenomic cell atlas of the aging mousebrain at the single-cell level. Single-cell DNA methylome and chromatinaccessibility datawillbegeneratedtoallowidentificationofcelltypesandcell-typespecificregulatoryelementsinthebrains of middle-aged (9 month old) and aged (18 month old) mice, and in brains of aged mice subject to caloric restriction. Aging-associated epigenomic signatures will be identified through comparison to single-cell epigenomicdatageneratedfromyoungmicegeneratedbyaBICCNU19CenterforEpigenomicsoftheMouse BrainAtlas (CEMBA). Through generationofa comprehensiveepigenome-basedbrain cell referenceatlas of theagingmousebrain,theproposedresearchwillprovideinvaluableresourcesfortheaging-researchfield.